The disclosed subject matter relates to techniques for superresolution microscopy, including techniques for deterministic emitter switch microscopy.
In certain conventional far-field optical microscopes, imaging resolution is limited to the diffraction limit, λ/2(n*sin(θ)), where λ is the illuminating light wavelength, n is the refractive index, and θ is the collection angle of the imaging optics. Generally speaking, the diffraction limit can be approximately half of the illuminating light's wavelength, or, e.g., approximately 200 nm in the visible spectrum.
In certain instances, it can be desirable to image at resolution below the diffraction limit. For example, as semiconductor device fabrication continues its trend toward increasingly smaller architecture, imaging techniques to resolve and inspect elements smaller than the diffraction limit can be useful for inspection or other purposes. Additionally, imaging for the biological sciences, such as imaging cell structures or certain proteins, can require imaging below the diffraction limit.
Certain techniques for imaging below the diffraction limit can generally be partitioned into two groups: (i) techniques to modify the fluorescence of a cluster of particles around an arbitrarily small area (for example in connection with stimulated emission depletion (STED), reversible saturable optical fluorescence transitions (RESOLFT), or saturated structured illumination microscopy (SSIM)), and (ii) techniques that rely on the stochastic switching of fluorescence molecules to reconstruct the positions of the molecules (for example in connection with stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), or fluorescence photoactivation localization microscopy (FPALM).
However, these techniques can require high excitation power, use of narrow spectrum light sources, particular fluorescent samples, expensive optical detection equipment, and intensive data processing techniques. For example, STED can require an excitation power higher than ˜GW/cm2. Moreover, techniques such as STED/RESOLFT can be limited to a small read out area for reasonable acquisition times (e.g., on the order of seconds) due to use of serial scanning imaging techniques rather than wide-field imaging. Techniques that rely on stochastic switching, for example, can require centroid fitting or other statistical processing of readouts over a period of time, which can inherently delay acquisition times due to the stochastic nature of the emitters. Moreover, certain fluorescent biomarkers used in connections with techniques for imaging below the diffraction limit can have brightness approximately an order of magnitude less than 105 counts/sec, can bleach, blink or degrade during excitation, and/or are toxic to cells.